Non-linking phosphorus backbone modifications are chemical changes made to the phosphodiester backbone of oligonucleotides to modify their stability, binding affinity, and resistance to enzymatic degradation without affecting the overall backbone linkage. These modifications introduce structural variations in the backbone phosphate groups of DNA or RNA without disrupting sequential linkages between nucleotides.
Incorporation of these modifications into the backbone of oligonucleotides enables the design of enhanced therapeutic oligonucleotides, such as antisense oligonucleotides, siRNAs, or aptamers, where increased stability and altered cellular uptake is desirable.
Kumar and Caruthers (2020) developed a series of novel oligonucleotides where one or both nonbridging oxygens in the phosphodiester backbone are replaced with an atom or molecule that introduces enhanced molecular properties beneficial for the development of therapeutic oligonucleotides with unique biological activity.
Kumar and Caruthers utilized two complementary approaches: Phosphoramidites that can act directly as synthons for the solid phase synthesis of oligonucleotide analogs. However, this approach was only sometimes feasible due to the instability of various synthons toward the reagents used during the synthesis of oligonucleotides. Therefore, the researchers selected a complementary approach to develop phosphoramidite synthons that allowed incorporation into oligonucleotides with minimum changes in the solid phase DNA synthesis protocols. This approach also enabled the introduction of functional groups for generating appropriate analogs post-synthetically. Oligonucleotides containing an alkyne group linked to phosphorus in the backbone allow attaching molecules such as amino acids and peptides.
| DNA Structure with Backbone | Modification replacing a nonbridging oxygen. |
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| X = S-; Phosphorothioate X = CH3 X = BH3-; Borane Phosphonate X = CH2COO-; Phosphonoacetate X = COO-; Phosphonoformate X = Triazoyl; Triazoylphosphonate X = OR; Phosphotriester X = NHR; Phosphoramidate X = BH2Py+; Pyridiniumboranephosphonates |
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Phosphorothioates (PS): In a PS modification, one of the non-bridging oxygen atoms in the phosphate is replaced with sulfur. This modification retains a negative charge but increases resistance to exonucleases and endonucleases, enhancing stability in biological systems.
Methylphosphonates: In this modification, one of the non-bridging oxygen atoms on the phosphate is replaced with a methyl group. This modification is neutral, as it lacks a negative charge, making it resistant to nucleases and improving cell permeability.
Borane phosphonates: Borane phosphonate is a chemical that involves replacing a non-bridging oxygen atom in the phosphate backbone with a borane group (BH₃).
Phosphonoacetates: Phosphonoacetates contain a phosphonate group (-PO₃²⁻) attached to an acetate moiety (-CH₂CO₂⁻) used in various chemical and biological contexts due to its unique structure and reactivity and solubility in water.
Phosphonoformates: Here the backbone is modified with a phosphonoformate group altering the properties of nucleic acids, influencing stability, charge, and biological activity of resulting oligonucleotides.
Triazoylphosphonates: In triazoylphosphonate modified oligonucleotides a triazole ring is linked to a phosphonate group. The 1,2,3-triazole ring is often synthesized via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry allowing the attachment of functional groups.
Phosphotriesters: Phosphotriesters can act as intermediates in synthetic and biological applications. The R group can be selected for specific chemical and biological properties.
Phosphoramidates: Here, an amino group replaces the non-bridging oxygen. This modification improves cellular delivery and binding characteristics, though it’s less commonly employed than other backbone modifications.
Pyridiniumboranephosphonates: In oligonucleotides modified with pyridiniumboranephosphonates a borane group (BH₃) is attached to a phosphonate moiety. These compounds are of interest in synthetic and medicinal chemistry due to their unique properties.
Reference
Kumar P, Caruthers MH. DNA Analogues Modified at the Nonlinking Positions of Phosphorus. Acc Chem Res. 2020 Oct 20;53(10):2152-2166. [Pubmed]
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